Use of GPR1 as target in preparing endometriosis-related diagnostic and / or therapeutic formulation

Abstract

A use of a GPR1 as a target in preparing an endometriosis-related diagnostic and / or therapeutic formulation, relating to the technical field of biomedicine. The present invention provides a use of a GPR1 target as a diagnostic marker in preparing a reagent or kit for diagnosing endometriosis. The endometriosis is selected from one of uterosacral ligament endometriosis, peritoneal endometriosis, rectovaginal endometriosis, deep infiltrating endometriosis, and ovarian endometriosis. The present invention discovers that the expression of the GPR1 is significantly increased in endometriosis tissues and cells, indicating that the GPR1 can be used as a diagnostic marker for endometriosis. It has been discovered in in-vitro and in-vivo models that GPR1 knockout significantly inhibits the abilities of hEM15A cells to proliferate, migrate, and invade and the growth of ectopic foci in animals, indicating that the GPR1 can be used as a new intervention target for clinically treating endometriosis.

Description

Application of GPR1 as a target in the preparation of diagnostic and / or therapeutic preparations related to endometriosis Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of GPR1 as a diagnostic marker and therapeutic target for endometriosis. Background Art

[0002] Endometriosis (abbreviated as "endometriosis") is a common gynecological disease that refers to the presence of endometrial-like lesions outside the uterine cavity. Numerous studies have linked endometriosis to a potential risk factor for a variety of conditions, including cancer, asthma, autoimmune diseases, and cardiovascular disease.

[0003] The diagnosis of endometriosis is currently a clinical challenge. Endometriosis is typically diagnosed based on clinical symptoms and signs, imaging studies, laparoscopy, and serum CA125 level testing. The universal gold standard is laparoscopic observation of the lesion morphology, combined with histopathological morphology for confirmation (endometrial glands and stroma are visible in the lesion, accompanied by inflammation and fibrosis). However, in some clinical cases, histopathological evidence is not available, and there are no clear molecular markers to assist with histopathological testing.

[0004] The treatment of endometriosis is mainly divided into surgical treatment and drug therapy. Surgical treatment is not very effective, and up to 75% of patients will relapse within 5 years after surgery; the purpose of drug treatment is to block the development of lesions. Common drugs are mainly divided into five categories: non-steroidal anti-inflammatory drugs, oral contraceptives, high-efficiency progestins, androgen derivatives, and gonadotropin-releasing hormone agonists. Because drug treatment mainly controls the hypothalamic-pituitary-ovarian reproductive endocrine axis and has strong side effects, it is not suitable for young patients with fertility needs. Even more regrettable is that there is still no ideal molecular target for the targeted treatment of endometriosis. Therefore, the discovery and functional identification of important molecular targets are the key to breaking the deadlock and will lay the molecular theoretical foundation for subsequent drug development.

[0005] G protein-coupled receptors (GPCRs) are a superfamily of seven-transmembrane receptors with over 1,000 members in the human genome. They are involved in regulating nearly all life processes, including perception, reproduction, development, and immunity, and play a crucial role in the development and progression of major diseases such as cancer and metabolism. GPCRs have long been a key target for drug development, with drugs targeting GPCRs accounting for nearly one-third of the global market. However, there are currently no non-hormonal GPCR-targeted drugs for the relief and treatment of endometriosis. In endometriosis research, antagonists or agonists targeting GPCRs, such as Neuropeptide S Receptor 1 and PGE2 Receptors, have been shown to significantly relieve pain and eliminate lesions in animal models, suggesting that targeting GPCRs holds great promise for the treatment of endometriosis.

[0006] G protein coupled receptor-1 (GPR1) belongs to Class A of GPCRs. In 1994, Marchese et al. reported cloning GPR1 from a hypothalamic cDNA library. It wasn't until 2008 that the orphan receptor GPR1 was "de-orphaned," with Barnea et al. using the Tango system discovering that Chemerin is a ligand for GPR1. In 2018, FAM19A1 was also identified as a ligand for GPR1. In 2019, a Cell article reported that osteocrin-2-19, cholecystokinin (CCK-33), and gastrin-releasing peptide were potential ligands for GPR1, but none of these three endogenous peptides had been confirmed at the biological function level.

[0007] Current understanding of GPR1 receptor signaling is primarily informed by the use of chemerin as an agonist. Chemerin is a widely distributed secreted protein with three primary receptors: CMKLR1 (Chemerin Chemokine-Like Receptor 1), GPR1, and CCRL2 (CC Motif Chemokine Receptor Like 2). Studies have shown that chemerin exhibits a high affinity for GPR1 (KD: 0.21 nM). Upon chemerin stimulation, GPR1 does not bind to any G protein but instead generates calcium ion fluctuations, further recruiting β-arrestin 1 and 2 and activating phosphorylated Erk1 / 2. Therefore, GPR1 operates in a G protein-independent, β-arrestin-biased signaling pathway under chemerin stimulation. The biological function of GPR1 remains largely unknown.

[0008] Summary of the Invention

[0009] Endometriosis is a common gynecological disease that is difficult to diagnose and incurable, and currently lacks an ideal diagnostic and therapeutic target. In response to the deficiencies in the prior art described above, the present invention aims to design and provide the use of GPR1 as a target in the preparation of diagnostic and / or therapeutic preparations related to endometriosis. The present invention is the first to discover that GPR1 expression is significantly elevated in endometriotic tissues and cells, suggesting that GPR1 can be used as a diagnostic marker for endometriosis. Further, in vitro and in vivo models, it was found that knocking out GPR1 significantly inhibited the proliferation, migration, and invasion of hEM15A cells, as well as the growth of ectopic lesions in animals, suggesting that GPR1 can serve as a new intervention target for the clinical treatment of endometriosis.

[0010] In order to achieve the above object, the present invention adopts the following technical solutions:

[0011] In a first aspect, the present invention provides the use of a GPR1 target as a diagnostic marker in the preparation of a reagent or kit for diagnosing endometriosis.

[0012] In the application, the endometriosis is selected from one of uterosacral ligament endometriosis, peritoneal endometriosis, rectovaginal endometriosis, deep infiltrating endometriosis, and ovarian endometriosis.

[0013] In a second aspect, the present invention provides use of a reagent for detecting GPR1 molecules in the preparation of a kit for diagnosing diseases caused by endometriosis.

[0014] In the application, the disease is selected from the group consisting of chronic pelvic pain caused by endometriosis, implantation failure, recurrent miscarriage, and infertility.

[0015] In the application, the reagent for detecting GPR1 molecules is selected from one of PCR primers, qPCR primers, FISH nucleic acid probes, antibodies, and binding polypeptides.

[0016] In a third aspect, the present invention provides use of an inhibitor of a GPR1 molecule in the preparation of a medicament for treating endometriosis.

[0017] In the application, the inhibitor is one of gRNA, dsRNA, siRNA, shRNA, microRNA or antisense RNA that targets GPR1 DNA or mRNA and inhibits gene transcription and protein expression.

[0018] In the application, the inhibitor is a biological or chemical agent that targets the protein sequence of the GPR1 molecule and produces an inhibitory or antagonistic effect on GPR1; the biological or chemical agent is selected from one of antibodies, polypeptides, and compounds.

[0019] In a fourth aspect, the present invention provides an endometriosis diagnostic kit comprising a GPR1 target as a diagnostic marker.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. Experiments in the present invention revealed that, compared with endometrial tissue in situ, GPR1 gene expression levels were significantly elevated in peritoneal ectopic lesions, uterosacral ligament ectopic lesions, rectovaginal lesions, deep infiltrating lesions, and ovarian ectopic lesions. qPCR and Western blot experiments confirmed that GPR1 gene and protein expression levels were significantly elevated in primary human ovarian endometriotic stromal cells. This suggests that GPR1 detection can be used to detect various types of endometriotic tissue and assist in pathological tissue identification.

[0022] 2. Experiments conducted in the present invention demonstrate that, using the hEM15A cell line, derived from the eutopic uterus of a patient with ovarian endometriosis, GPR1 knockout cell lines were constructed using the CRISPR Cas9 system. GPR1 knockout was found to significantly inhibit cell proliferation, migration, and invasion. An endometriosis animal model was established using GPR1 knockout mice, which revealed significant regression of endometriotic lesions. Inhibiting GPR1 expression can inhibit the biological functions of endometrial stromal cells and the growth of lesions in vivo, suggesting that GPR1 may serve as a therapeutic target for endometriosis, and its inhibitors may be used to develop drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 shows the analysis results of the dataset numbered GSE141549 in the GEO database;

[0024] Figure 2 is a graph showing the results of a Western blot experiment, wherein A is a graph showing the qPCR primer sequence for GPR1; B is a graph showing the expression level of the GPR1 gene; and C is a graph showing the expression level of the GPR1 protein.

[0025] Figure 3 shows the gRNA sequence, qPCR experiment, Western blot experiment results, and Transwell migration and invasion assay results;

[0026] Figure 4 shows the results of lesion construction in model mice using the allogeneic surgical induction method. DETAILED DESCRIPTION

[0027] The following embodiments are merely some of the embodiments of the present invention, rather than all of them. Therefore, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the claimed invention, but rather merely represents selected embodiments of the present invention. All other embodiments derived by those skilled in the art based on the embodiments of the present invention without creative effort are intended to fall within the scope of protection of the present invention.

[0028] In the following examples, the reagents, instruments, equipment, preparation methods or processes are all commonly used techniques in the art unless otherwise specified.

[0029] Example 1:

[0030] We retrieved the dataset GSE141549 from the Gene Expression Omnibus (GEO, https: / / www.ncbi.nlm.nih.gov / geo / ) and downloaded the file GSE141549_batch Corrected normalized Arrays combined.xlsx to extract GPR1 gene expression data. Results As shown in Figure 1, compared with eutopic uterine tissue from healthy women (Figure 1-A, n = 38), GPR1 gene expression levels were significantly increased in ectopic lesions of the peritoneum (Figure 1-B, n = 76), ectopic lesions of the uterosacral ligament (Figure 1-C, n = 26), rectovaginal lesions (Figure 1-D, n = 22), deep infiltrating lesions (Figure 1-E, n = 40), and ectopic lesions of the ovary (Figure 1-F, n = 28).

[0031] Example 2:

[0032] 1. Tissue acquisition and primary cell extraction: Tissues were obtained from Shenzhen University General Hospital. All patients provided written informed consent, and the procedures were approved by the Ethics Committee of Shenzhen University General Hospital and the Ethics Committee of Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences.

[0033] After the tissue was obtained, it was manipulated in a sterile table and minced with sterile ophthalmic scissors. Digestive solution (DNase I and collagenase) was added and incubated in a 37°C water bath for 1.5 h. The tissue was shaken for 10 s every 10 min until digestion. The tissue was filtered through a 40 μm cell sieve and placed in a cell incubator for culture.

[0034] 2. Extraction of cell RNA: discard the culture medium of the cells, wash three times with PBS, aspirate 1ml of RNA lysis solution, pipette the bottom of the dish, aspirate all the cell lysate into an RNase-free EP tube, and let it stand on the ice box for 5 minutes. Add 200μL of chloroform to the tube and let it stand in the ice box for 5 minutes. Centrifuge at 4℃, 12000rpm, 10min (take a new EP tube and place it on ice to pre-cool). Aspirate the upper aqueous phase into a new EP tube, add 1000μL of isopropanol, and mix manually. Centrifuge at 4℃, 12000rpm, 10min. Discard the supernatant, add 1mL of 75% ethanol to the tube, centrifuge at 4℃, 12000rpm, 5min. Discard the supernatant and dry it to obtain RNA.

[0035] 3. cDNA reverse transcription: Prepare the reverse transcription reaction solution according to the components given in the instructions. Mix gently and incubate at 37°C for 15 minutes, 85°C for 5 seconds, and then at 4°C to obtain cDNA.

[0036] 4.qPCR: Primers were synthesized by Shanghai Bioengineering. The reaction solution was prepared according to the components specified in the instructions, and the Ct value was obtained using the two-step PCR conditions. The relative expression content was calculated according to calculate.

[0037] 5. Cell protein extraction: discard the culture medium from the cells, wash three times with PBS, add protein lysis buffer containing protease inhibitors, scrape the cells with a scraper and place on ice, shake once every 10 minutes, centrifuge at 12000 rpm at 4℃ after 30 minutes, take the supernatant and place it in a new EP tube for protein quantification.

[0038] 6. Western blot: SDS-PAGE electrophoresis was performed according to the amount of protein per well of 30 micrograms. After the electrophoresis, the protein was transferred to a nitrocellulose membrane at 250 mA for 2 hours and then blocked with 5% skim milk powder. After 2 hours at room temperature, GPR1 antibody (purchased from abcam, catalog number: ab249312) was prepared at a ratio of 1 / 1000, incubated with NC membrane, and placed in a 4°C refrigerator overnight. The next day, the NC membrane was eluted with TBST solution, once for 10 minutes, and three times with a secondary antibody at a ratio of 1 / 3000, incubated with NC membrane, and placed at room temperature for 1 hour. After elution with TBST, the NC membrane was covered with ECL to obtain the results. As shown in Figure 2, the qPCR primer sequence of GPR1 is Figure 2A. qPCR results using this primer as a probe showed that GPR1 gene expression was significantly increased in ectopic endometrial stromal cells (Ovarian Endometriosis-Ecpotic, abbreviated as OvEM-Ec) from primary endometriosis patients (ovarian endometriosis) compared to primary healthy eutopic endometrial stromal cells (Normal-Eupotic, abbreviated as Nor-Eu) and eutopic endometrial stromal cells (Ovarian Endometriosis-Eupotic, abbreviated as OvEM-Eu), as shown in Figure 2B. Furthermore, GPR1 protein expression was significantly increased in OvEM-Ec compared to Nor-Eu and OVEM-Eu, as shown in Figure 2C.

[0039] Example 3:

[0040] 1. GPR1 expression knockout: A three-plasmid lentiviral system (pHBLV-U6-gRNA-EF1-CAS9-PURO vector, psPAX2 vector, and pMD2G vector) was used to construct lentivirus. After concentrating the lentivirus, hEM15A cells were infected. After 24 hours, 2 μg / mL puromycin was added for selection, and then single-cell clones were screened using the limiting dilution method. qPCR and Western blot results showed that GPR1 was successfully knocked out in hEM15A cells. The sequence information of the gRNA is shown in Figure 3A. As shown in Figures 3B and C, CRISPR Cas9 gene editing was performed with this gRNA to knock out GPR1 in hEM15A cells. qPCR and Western blot results showed that the gene and protein expression of GPR1 were knocked out in two cell clones.

[0041] 2. Cell Proliferation Assay: hEM15A cells were plated at 2000 cells / well in a 96-well plate (culture medium with 10% serum concentration) and assayed after 24, 48, 72, and 96 hours. The assay method involved aspirating the culture medium and adding CCK-8 reagent (purchased from Beyotime) at a 1 / 10 volume ratio (100 μL per well) to serum-free culture medium. Cell viability was assessed by reading absorbance at 450 nm using a microplate reader. The results, shown in Figure 3D, indicate that the proliferation of the GPR1 knockout cell line was significantly inhibited by the CCK8 assay.

[0042] 3. Cell migration ability: Using Transwell chambers (purchased from Corning) as a research model, hEM15A cells were placed in serum-free culture medium at 100,000 / well and added to the upper layer of the chamber. Culture medium containing 10% serum was added to the lower layer of the chamber as a chemoattractant. After 24 hours, the chambers were removed, and the cells that migrated from under the chamber membrane were fixed and stained with methanol and crystal violet, respectively. The non-migrated cells on the upper side were then wiped away with a cotton swab, photographed, and the migration rate was calculated. The results, as shown in Figure 3E, show a significant decrease in migration of the GPR1 knockout cell line.

[0043] 4. Cell Invasion Ability: Transwell chambers were used as a research model. 50 μL of Metrial Gel (mixed with serum-free medium at a 1 / 10 volume ratio per well, purchased from Corning) was pre-coated on the upper chamber layer and incubated at 37°C for half an hour. hEM15A cells were plated at 100,000 cells / well in serum-free medium and added to the upper chamber layer. Medium containing 10% serum was added to the lower chamber layer as a chemoattractant. After 48 hours, the chambers were removed and cells invading from the lower chamber membrane were fixed and stained with methanol and crystal violet, respectively. Non-invading cells on the upper side of the membrane were removed with a cotton swab, photographed, and the invasion rate was calculated. As shown in Figure 3F, the invasion ability of the GPR1 knockout cell line was significantly reduced.

[0044] Example 4:

[0045] The peritoneal endometriosis mouse model (PeEM model) is a widely accepted model in endometriosis research and is widely used to analyze the functional effects of specific genes and compounds on endometriotic lesions. PeEM models are categorized by surgical or injection-induced modeling method, and by endometrial origin as autologous or allogeneic.

[0046] This invention uses an allogeneic surgical induction method. The model has a high success rate and strong reproducibility. Compared with the sham group, PeEM mice show reduced fertility and increased pain response, which better reflects clinical symptoms. The construction process is as follows:

[0047] 1. Place 7-week-old C57BL / 6J female mice in the animal room and conduct the experiment one week later;

[0048] 2. Both donor and recipient mice are in estrus as the model construction objects;

[0049] 3. Kill the donor mouse by cervical dislocation, remove the uterus, and remove the ovaries, fallopian tubes, blood vessels, and adipose tissue;

[0050] 4. Divide the uterus into two halves and cut them open longitudinally with surgical scissors to expose the endometrial layer;

[0051] 5. Use a scalpel to divide the two uterine sections into four equal pieces and place them in normal saline;

[0052] 6. Anesthetize the recipient mice with 2.5% Avertin (intraperitoneal injection, 300 μl);

[0053] 7. Open the abdominal cavity along the midline;

[0054] 8. Use 6-0 absorbable surgical sutures to fix the four uterine pieces to the sides of the abdominal wall (two pieces / side, with the endometrial surface close to the abdominal wall);

[0055] 9. Suture the muscles and skin, disinfect, observe every other day, and terminate after four weeks. Collect samples, weigh and count the lesions. The results are shown in Figure 4. - / - One side of the uterus of WT mice (divided into four equal parts) was surgically transplanted into the abdominal wall of WT mice to construct WT→WT and Gpr1 - / - →WT mice, and the mice were killed one month after the construction, and the abdominal wall lesions of each mouse were collected, as shown in Figure 4A. The lesions on the abdominal wall were peeled off and weighed. The results showed that compared with the WT→WT group, Gpr1 - / - The weight of the lesions in the WT group was significantly reduced (Figure 4B). In addition, the statistical results showed that about 8.8% of the lesions in the WT→WT group subsided, while the Gpr1 - / - → Approximately 72.5% of the lesions in the WT group regressed (Figure 4C).

[0056] The above description is only a preferred embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent transformation made using the contents of the present invention description and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Use of GPR1 as a target in the preparation of diagnostic and / or therapeutic preparations for endometriosis-related diseases.

2. Application of GPR1 target as a diagnostic marker in the preparation of reagents or kits for diagnosing endometriosis.

3. The use according to claim 2, characterized in that The endometriosis is selected from one of uterosacral ligament endometriosis, peritoneal endometriosis, rectovaginal endometriosis, deep infiltrating endometriosis and ovarian endometriosis.

4. Use of a reagent for detecting GPR1 molecules in the preparation of a kit for diagnosing diseases caused by endometriosis.

5. The use according to claim 4, characterized in that The disease is selected from the group consisting of chronic pelvic pain caused by endometriosis, implantation failure, recurrent miscarriage, and infertility.

6. The use according to claim 4, characterized in that The reagent for detecting the GPR1 molecule is selected from one of PCR primers, qPCR primers, FISH nucleic acid probes, antibodies, and binding polypeptides.

7. Use of inhibitors of GPR1 molecules in the preparation of drugs for the treatment of endometriosis.

8. The use according to claim 7, characterized in that The inhibitor is one of gRNA, dsRNA, siRNA, shRNA, microRNA or antisense RNA that targets the DNA or mRNA of GPR1 and inhibits gene transcription and protein expression.

9. The use according to claim 7, characterized in that The inhibitor is a biological or chemical agent that targets the protein sequence of the GPR1 molecule and produces an inhibitory or antagonistic effect on GPR1; the biological or chemical agent is selected from one of antibodies, polypeptides, and compounds.

10. A diagnostic kit for endometriosis, characterized in that: The GPR1 target was included as a diagnostic marker.

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